Project Summary: Cellular molecular mismatch repair (MMR) machinery is charged with scanning and repairing DNA as it is continuously bombarded by a variety of stresses. Mutations in human mismatch recognition proteins have been implicated in over half of hereditary non-polyposis colorectal cancers and -15% of endodermal and ovarian cancers in humans. Despite the urgent need to understand how MMR proteins recognize damaged DNA, there is currently little agreement about the molecular mechanism of mismatch damage recognition. This proposal outlines a series of experiments that combine single-molecule biophysics methodologies with ID-diffusion assays to solve a long-standing debate in the DNA repair field: How do mismatch repair proteins locate and respond to mispaired bases? To accomplish this, total internal reflection fluorescence microscopy (TIRFM) will be employed to monitor individual fluorescently labeled MMR proteins bound to arrays of DNA molecules that are tethered to the passivated surface of a microfluidic sample chamber. The TIRFM technique will facilitate direct visualization of the E. coli proteins MutS and MutL, which together facilitate the first steps in DNA mismatch recognition. MutS and MutL homologues are found in nearly all organisms, including humans. Initial studies will observe the ATP-dependent MutS-DNA proofreading mechanism, and will subsequently focus to the role of MutS-MutL complexes in triggering downstream repair events. The role of ATP hydrolysis by MMR proteins in binding, proofreading, and subsequently locating a mismatch site will be probed by a combination of site-directed protein mutagenesis and studies on non-hydrolysable ATP analogs. I Relevance: Many common human cancers occur when an erroneously mispaired segment of duplex DNA is not repaired by the molecular mismatch machinery. This research aims to unravel the complicated sequence of molecular events that allow damaged DNA to be recognized and ultimately repaired by mismatch repair proteins.